Genomic, Epigenomic, and Transcriptional Characterisation of Carbapenem and Colistin Resistance Mechanisms inKlebsiella pneumoniaeandEnterobacterspecies

Abstract

The emergence of colistin and carbapenem-resistantKlebsiella pneumoniaeisolates presents a significant global health threat. This study investigates the resistance mechanisms in sixK. pneumoniaeand fourEnterobactersp. isolates lacking carbapenemases ormcrgenes using genomics and transcriptomics. The ten isolates were classified into three categories: non-carbapenemase-producing, carbapenem-resistant strains (n = 4), non-mcr–producing colistin-resistant strains (n = 5), and one isolate susceptible to both antibiotics.The analysis included phenotypic characterization using MicroScan ID/AST, enzyme (MCR and Metallo β-lactamase) and efflux pump inhibition (EPI) assays. Whole-genome sequencing, RNA sequencing, and bioinformatics tools were employed in subsequent analysis. Most of theK. pneumoniaewere ST307 with KL102 and O1/O2V2 serotypes. MicroScan revealed multidrug resistance, and AMR analysis identified numerous ARGs inK. pneumoniae.Enterobacterspecies possessed fewer resistance genes; nevertheless, they encoded virulence factors and gene mutations, potentially impacting the AST profile.K. pneumoniaeARGs were mainly plasmid-borne, with IncFIB(K)/IncFII(K) in Kp_15 harbouring up to nineteen ARGs. Virulence factors included biofilm formation, capsule production, and type IV secretion. Epigenomic investigations revealed prevalent type I (M1.Ecl34977I) and type II (M.Kpn34618Dcm) restriction modification sites. Compared to international isolates, the study isolates phylogenetically clustered more closely with Chinese strains. Transcriptomics showed high efflux pump activity in carbapenem-resistant isolates, confirmed by EPI. Further, mutations were identified in outer membrane proteins. Colistin-resistant isolates exhibited high capsule production, efflux pump, and putative glycotransferase activity, potentially influencing their phenotypes.In conclusion, genomic and transcriptional analyses enhanced our understanding of adaptive mechanisms in clinical multidrug-resistant pathogens, posing significant public health challenges.